The inherently flexibility of the sugar moiety of conventional nucleosides has been arrested by replacing the sugar moiety with a rigid carbocyclic ring (i.e., bicyclo[3.1.0]hexane) that mimics either the North (N) or South (S) conformation of conventional nucleosides in the pseudorotational cycle. Our main objective has been to systematically probe a series of nucleoside/nucleotide binding enzymes with sets of conformationally rigid N and S substrates to learn about the preferred mode of binding. A secondary objective has been to construct modified nucleic acids that incorporate some of these fixed units to either reinforce or disrupt the typical B- or A-DNA conformations associated with S and N conformations, respectively. The corresponding 5'-triphosphates of the locked antipodes of adenosine, N and S methanocarba adenosines (N-MCA and S-MCA), were synthesized and tested as substrates for HIV-RT. Since these compounds have a 3'-OH group, they allow chain elongation. Therefore, any effect on chain termination of DNA synthesis by these compounds has to be related to a distant distortion effect induced by their locked conformation, following incorporation. Using a template where we varied the number of adjacent T residues (1 through 4), we studied the ability of HIV RT to reach full length DNA as a function of the number of N-MCA or S-MCA residues incorporated. The results showed that for a template with 1-2 adjacent T residues, N-MCA termination of DNA synthesis occurred mainly 7-9 nt from the site of incorporation, while with S-MCA it occurred 9-11 nt from the site of incorporation. In both cases some full-length product was obtained. However, as the number of adjacent T residues increased (3-4), N-MCA was able to correct the distortion and only full length DNA was obtained. On the other hand, 3-4 T residues in the case of S-MCA proved fatal and no full length DNA was obtained. A novel approach for the synthesis of North conformers that involved a lipase-catalyzed resolution step following the formation of the bicyclo[3.1.0]hexane system was very successful and has guaranteed an ample supply of enatiomerically pure compounds. The same approach is being investigated for the South analogues. We have synthesized a series of short oligodeoxynucleotides (ODNs) corrresponding to the self-complementary EcoR1 recognition sequence [ds(5'-CGCGAATTCGCG-3')] where the middle A's and the middle T's have been replaced by locked adenosines and locked thymidines. These sequences are the subject of current NMR, CD and crystallographic studies.